Mycophenolic acid (MPA) is a potent and specific inhibitor of mammalian inosine-monophosphate dehydrogenases (IMPDH); most microbial IMPDHs are not sensitive to MPA. MPA-resistant mutants of human IMPDH type II were isolated in order to identify the structural features that determine the species selectivity of MPA. Three mutant IMPDHs were identified with decreased affinity for MPA The mutation of Gln277 --> Arg causes a 9-fold increase in the Ki of MPA, a 5-6-fold increase in the Km values for IMP and NAD, and a 3-fold decrease in kcat relative to wild type. The mutation of Ala462 --> Thr causes a 3-fold increase in the Ki for MPA, a 2.5-fold increase in the Km for NAD, and a 1.5-fold increase in kcat. The combination of these two mutations does not increase the Ki for MPA, but does increase the Km for NAD 3-fold relative to Q277R and restores kcat to wild type levels. Q277R/A462T is the first human IMPDH mutant with increased Ki for MPA and wild type activity. The third mutant IMPDH contains two mutations, Phe465 --> Ser and Asp470 --> Gly. Ki for MPA is increased 3-fold in this mutant enzyme, and Km for IMP is also increased 3-fold, while the Km for NAD and kcat are unchanged. Thus increases in the Ki for MPA do not correlate with changes in Km for either IMP or NAD, nor to changes in kcat. All four of these mutations are in regions of the IMPDH that differ in mammalian and microbial enzymes, and thus can be structural determinants of MPA selectivity.